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Subtype-independent near full-length HIV-1 genome sequencing and assembly to be used in large molecular epidemiological studies and clinical management.

Grossmann S, Nowak P, Neogi U - J Int AIDS Soc (2015)

Bottom Line: It also enables genotypic resistance testing (GRT) for all drug target sites allowing effective intervention strategies for control and prevention in high-risk population groups.It will facilitate the understanding of the HIV-1 pandemic population dynamics and outline effective intervention strategies.Furthermore, it can potentially be applicable in clinical management of drug resistance by evaluating DRMs against all available antiretrovirals in a single assay.

View Article: PubMed Central - PubMed

Affiliation: Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden.

ABSTRACT

Introduction: HIV-1 near full-length genome (HIV-NFLG) sequencing from plasma is an attractive multidimensional tool to apply in large-scale population-based molecular epidemiological studies. It also enables genotypic resistance testing (GRT) for all drug target sites allowing effective intervention strategies for control and prevention in high-risk population groups. Thus, the main objective of this study was to develop a simplified subtype-independent, cost- and labour-efficient HIV-NFLG protocol that can be used in clinical management as well as in molecular epidemiological studies.

Methods: Plasma samples (n=30) were obtained from HIV-1B (n=10), HIV-1C (n=10), CRF01_AE (n=5) and CRF01_AG (n=5) infected individuals with minimum viral load >1120 copies/ml. The amplification was performed with two large amplicons of 5.5 kb and 3.7 kb, sequenced with 17 primers to obtain HIV-NFLG. GRT was validated against ViroSeq™ HIV-1 Genotyping System.

Results: After excluding four plasma samples with low-quality RNA, a total of 26 samples were attempted. Among them, NFLG was obtained from 24 (92%) samples with the lowest viral load being 3000 copies/ml. High (>99%) concordance was observed between HIV-NFLG and ViroSeq™ when determining the drug resistance mutations (DRMs). The N384I connection mutation was additionally detected by NFLG in two samples.

Conclusions: Our high efficiency subtype-independent HIV-NFLG is a simple and promising approach to be used in large-scale molecular epidemiological studies. It will facilitate the understanding of the HIV-1 pandemic population dynamics and outline effective intervention strategies. Furthermore, it can potentially be applicable in clinical management of drug resistance by evaluating DRMs against all available antiretrovirals in a single assay.

No MeSH data available.


Related in: MedlinePlus

Phylogenetic and variability analysis of sequenced Swedish HIV-1 strains. (a) Maximum likelihood phylogenetic tree with reference HIV-1 sequences downloaded from Los Alamos Database. Four subtypes are indicated: HIV-1B (dark blue), HIV-1C (orange), CRF01_AG (green) and CRF02_AG (purple). The Swedish strains are indicated with filled circle with a respective colour. (b) Genetic diversity of HIV-1 subtypes: all the 24 HIV-1 genomes were aligned with reference to HXB2 [GenBank:K03455]. For each sequence, every nucleotide differing from the reference HXB2 strain (mutation) is shown as a green line, an insertion is shown in orange, and a deletion is shown in purple. The top panel shows the open-reading frame of HIV-1 genes: gag (violet), pol (lemon green), vif (pink), vpr (light red), tat exon 1 and 2 (black), rev exon 1 and 2 (grey), vpu (cyan), env (light yellow) and nef (green).
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Figure 0002: Phylogenetic and variability analysis of sequenced Swedish HIV-1 strains. (a) Maximum likelihood phylogenetic tree with reference HIV-1 sequences downloaded from Los Alamos Database. Four subtypes are indicated: HIV-1B (dark blue), HIV-1C (orange), CRF01_AG (green) and CRF02_AG (purple). The Swedish strains are indicated with filled circle with a respective colour. (b) Genetic diversity of HIV-1 subtypes: all the 24 HIV-1 genomes were aligned with reference to HXB2 [GenBank:K03455]. For each sequence, every nucleotide differing from the reference HXB2 strain (mutation) is shown as a green line, an insertion is shown in orange, and a deletion is shown in purple. The top panel shows the open-reading frame of HIV-1 genes: gag (violet), pol (lemon green), vif (pink), vpr (light red), tat exon 1 and 2 (black), rev exon 1 and 2 (grey), vpu (cyan), env (light yellow) and nef (green).

Mentions: The phylogenetic analysis identified the same subtype as identified by the pol region with 10 as HIV-1C, 8 as HIV-1B and 3 each as 01_AE and 02_AG (Figure 2a). The sequence variability of the 24 samples compared to HXB2 sequence is presented in Figure 2b. This indicates higher sequence variability in the env region and the subtype-specific signatures over the genome specifically in the Gag-p6 region.


Subtype-independent near full-length HIV-1 genome sequencing and assembly to be used in large molecular epidemiological studies and clinical management.

Grossmann S, Nowak P, Neogi U - J Int AIDS Soc (2015)

Phylogenetic and variability analysis of sequenced Swedish HIV-1 strains. (a) Maximum likelihood phylogenetic tree with reference HIV-1 sequences downloaded from Los Alamos Database. Four subtypes are indicated: HIV-1B (dark blue), HIV-1C (orange), CRF01_AG (green) and CRF02_AG (purple). The Swedish strains are indicated with filled circle with a respective colour. (b) Genetic diversity of HIV-1 subtypes: all the 24 HIV-1 genomes were aligned with reference to HXB2 [GenBank:K03455]. For each sequence, every nucleotide differing from the reference HXB2 strain (mutation) is shown as a green line, an insertion is shown in orange, and a deletion is shown in purple. The top panel shows the open-reading frame of HIV-1 genes: gag (violet), pol (lemon green), vif (pink), vpr (light red), tat exon 1 and 2 (black), rev exon 1 and 2 (grey), vpu (cyan), env (light yellow) and nef (green).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482814&req=5

Figure 0002: Phylogenetic and variability analysis of sequenced Swedish HIV-1 strains. (a) Maximum likelihood phylogenetic tree with reference HIV-1 sequences downloaded from Los Alamos Database. Four subtypes are indicated: HIV-1B (dark blue), HIV-1C (orange), CRF01_AG (green) and CRF02_AG (purple). The Swedish strains are indicated with filled circle with a respective colour. (b) Genetic diversity of HIV-1 subtypes: all the 24 HIV-1 genomes were aligned with reference to HXB2 [GenBank:K03455]. For each sequence, every nucleotide differing from the reference HXB2 strain (mutation) is shown as a green line, an insertion is shown in orange, and a deletion is shown in purple. The top panel shows the open-reading frame of HIV-1 genes: gag (violet), pol (lemon green), vif (pink), vpr (light red), tat exon 1 and 2 (black), rev exon 1 and 2 (grey), vpu (cyan), env (light yellow) and nef (green).
Mentions: The phylogenetic analysis identified the same subtype as identified by the pol region with 10 as HIV-1C, 8 as HIV-1B and 3 each as 01_AE and 02_AG (Figure 2a). The sequence variability of the 24 samples compared to HXB2 sequence is presented in Figure 2b. This indicates higher sequence variability in the env region and the subtype-specific signatures over the genome specifically in the Gag-p6 region.

Bottom Line: It also enables genotypic resistance testing (GRT) for all drug target sites allowing effective intervention strategies for control and prevention in high-risk population groups.It will facilitate the understanding of the HIV-1 pandemic population dynamics and outline effective intervention strategies.Furthermore, it can potentially be applicable in clinical management of drug resistance by evaluating DRMs against all available antiretrovirals in a single assay.

View Article: PubMed Central - PubMed

Affiliation: Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden.

ABSTRACT

Introduction: HIV-1 near full-length genome (HIV-NFLG) sequencing from plasma is an attractive multidimensional tool to apply in large-scale population-based molecular epidemiological studies. It also enables genotypic resistance testing (GRT) for all drug target sites allowing effective intervention strategies for control and prevention in high-risk population groups. Thus, the main objective of this study was to develop a simplified subtype-independent, cost- and labour-efficient HIV-NFLG protocol that can be used in clinical management as well as in molecular epidemiological studies.

Methods: Plasma samples (n=30) were obtained from HIV-1B (n=10), HIV-1C (n=10), CRF01_AE (n=5) and CRF01_AG (n=5) infected individuals with minimum viral load >1120 copies/ml. The amplification was performed with two large amplicons of 5.5 kb and 3.7 kb, sequenced with 17 primers to obtain HIV-NFLG. GRT was validated against ViroSeq™ HIV-1 Genotyping System.

Results: After excluding four plasma samples with low-quality RNA, a total of 26 samples were attempted. Among them, NFLG was obtained from 24 (92%) samples with the lowest viral load being 3000 copies/ml. High (>99%) concordance was observed between HIV-NFLG and ViroSeq™ when determining the drug resistance mutations (DRMs). The N384I connection mutation was additionally detected by NFLG in two samples.

Conclusions: Our high efficiency subtype-independent HIV-NFLG is a simple and promising approach to be used in large-scale molecular epidemiological studies. It will facilitate the understanding of the HIV-1 pandemic population dynamics and outline effective intervention strategies. Furthermore, it can potentially be applicable in clinical management of drug resistance by evaluating DRMs against all available antiretrovirals in a single assay.

No MeSH data available.


Related in: MedlinePlus